3. RESULTS AND DISCUSSION
3.2 RESVERATROL IN COMBINATION WITH SKI II, PDMP AND MYRIOCIN AFFECT THE
Progression of SD1 and SUP-B15 Ph + ALL Cells
To understand the potential mechanisms behind the growth suppressive effects of resveratrol, SPT, SK and GCS inhibitors, resveratrol: SPT inhibitor, resveratrol: SK inhibitor and resveratrol: GCS inhibitor combinations on SD1 and SUP-B15 cells, the distributions of the cells in the cell cycle phases were determined by flow cytometry via Propidium Iodide (PI) staining. 20 μM resveratrol was combined with 2.5 μM SKI II, 10 μM PDMP and 100 nM Myriocin for SD1 cells. Treatment of SD1 cells with 20 μM resveratrol resulted in decreases in cell population at G0 / G1 phase whereas led to increases at S phase as compared to control cells. The percentage of cell population at the G0/
G1 phase was 57.03% and 33.9% while it was 5.7% and 6.5% at the S phase for control and 20 μM resveratrol, respectively. 2.5 μM SKI II treatment reduced the percentage of cells at G0 /G1 phase (36.1%) as compared to the control (57.3%) while increased it at S (7.5%) and very slightly at G2 /M (7.9%) (Control: S: 5.7% and G2 / M: 7.6%). The percentage of cell population at G0 /G1 phase and S phase were as 33.6% and 10% after 20 μM resveratrol and 2.5 μM SKI II treatment and S phase population was found to be increased as compared to 20 μM resveratrol. Thus, the combination treatment led to cell cycle arrest at S phase. This result supported the data shown and described in
46
Figure 3.1.3.a. Similarly, 20 μM resveratrol + 10 μM PDMP combination arrested the cells at S phase. The percentage of cell population for this combination was 13.7% (Control: 5.7%, 20 μM resveratrol: 6.5%, 10 μM PDMP: 6.8%) (Figure 3.2.1.b). Thus, there is an arrest in the S phase of the cell cycle as a result of the combination treatment. These results support cell proliferation data (Figure 3.1.3.b) and, the distribution of SD1 cells treated with 20 μM resveratrol at G0 / G1, S and G2 / M phases was 33.9%, 6.5%, and 3.1%
while it was 57.03%, 5.7% and 7.6% for control cells, respectively. As a result of 100 nM myriocin treatment, the distribution of cells in G0 / G1, S and G2 / M phases was 70%, 6.5% and 3.2%, respectively. 100 nM myriocin treatment resulted in the distribution of cells at G0 / G1, S and G2 / M phases as 70%, 6.5% and 3.2%, respectively. The distribution of cells at G0 / G1, S and G2 / M phases were 46%, 9.3%, and 4.7%, respectively after 20 μM resveratrol + 100 nM myriocin treatment. Based on these results, 20 μM resveratrol + 100 nM myriocin was found to be effective at S phase.
47
Figure 3.2.1. Cell cycle distrubution of SD1 cells after treatment with Resveratrol, SKI II, PDMP, Myriocin, Resveratrol: SK-1 Inhibitor (a), Resveratrol: GCS inhibitor (b) and Resveratrol: SPT Inhibitor (c) combinations. The results derived from the means of three independent experiments are represented as mean± SE
48
Treatment of SUP-B15 cells with 5- and 10 μM resveratrol, 1 μM SKI II resulted in decreases in cell population at G0 / G1 phase as compared to control cells, while it increased the cell population at S phase. Distribution of the cells were 53.95%, 45.9% and 42.4% at G0 / G1 phase while S phase cell population was 15.4%, 16.35% and 17.35% for control, 5- and 10 μM resveratrol, respectively (Figure 3.2.2.a). 1 μM SKI II decreased the percentage of cells at G0 / G1 phase (43.8%) while increasing at S (17%) and G2 / M (19.3%) phases compared to the control (G0/G1: 53.95%, S: 15.4% and G2 / M:
17%). 5- and 10 μM resveratrol in combination with 1 μM SKI II synergistically led to decreases at G0 / G1 phase as 41% and 27.3%, respectively (as compared to defined concentrations of resveratrol). Cell populations at G2 / M phase were found to be 17.9% and 3.5% for the combination of 5- and 10 μM resveratrol with 1 μM SKI II, respectively. (G2 / M phase cell population were 19.6% and 17.55% for 5- and 10 μM resveratrol, respectively). On the other hand, 5- and 10 μM resveratrol with 1 μM SKI II arrested the cells at S phase (20.3% and 27%, respectively) compared to defined concentrations of resveratrol. Thus, while combination treatments reduced the percentages of SUP-B15 cells at G0 / G1 and G2 / M phases, the cells were arrested at S phase and the most effective combination was found to be 10 μM resveratrol + 1 μM SKI II (Figure 3.2.2.a).
This result was in accordance with the result shown and described in Figure 3.1.4.a. 5- and 10 μM resveratrol + 1 μM PDMP treatment decreased the cell population at G0 / G1 as 44% and 39.7%, respectively (Control: 53.95%, 5 μM resveratrol: 45.9%, 10 μM resveratrol: 42.4%, 1 μM PDMP 49.8%). At S phase, the cell population distribution were 18.5% and 21.3% for 5- and 10 μM resveratrol + 1 μM PDMP combinations, respectively (Control: 15.4%, 5 μM resveratrol: 16.35%, 10 μM resveratrol: 17.35%, PDMP 1 μM: 16.2%) (Figure 3.2.2.b). Thus, the combination treatment resulted in S phase arrest which supported the cell proliferation data (Figure 3.1.4.b). Based on these results, it is proved that combinations of resveratrol with SKI II and PDMP suppress cell proliferation by arresting the cell at S phase and inhibiting DNA synthesis.
49
The distributions of SUP-B15 cells at G0 /G1, S and G2 / M phases as shown in Figure 3.2.2.a after treatment with 5- and 10 μM resveratrol were 45.9%, 16.35%, 19.6% and 42.4%, 17.35% and %17.55 respectively. For control cells, it was 53.95%, 15.4% and 17%, respectively. The distribution of the cells at G0 / G1, S and G2 / M phases after 100 nM myriocin treatment was 49.5%, 12.7% and 22.4%, respectively. Myriocin caused arrest at G2 / M phase compared to control cells while it decreased the cell population at G0 / G1 and S phases. For the 5- and 10 μM resveratrol + 100 nM myriocin combinations, the distribution of cells at G0 / G1, S and G2 / M phases were 44.1%, 12.2%, 19.1%
and 38.3%, 14%, 20.4%, respectively (Figure 3.2.2.c). For both combinations, the cell population was reduced at G0 / G1 phase. Based on these results, the combination of 5- and 10 μM resveratrol with 100 nM myriocin did not appear to be effective on cell cycle progresion, which supported the proliferation data (Figure 3.1.4.c).
50
Figure 3.2.2 Cell cycle distrubution of SUP-B15 cells after treatment with Resveratrol, SKI II, PDMP, Myriocin, Resveratrol: SK-1 Inhibitor (a), Resveratrol: GCS inhibitor (b) and Resveratrol: SPT Inhibitor (c) combinations. The results derived from the means of three independent experiments are represented as mean± SE
51
The reason why inhibitors, resveratrol and resveratrol-inhibitor combinations suppressed the cell proliferation in SD1 and SUP-B15 cells can be explained with the effects of them on cell cycle progression. Based on the cell cycle data for SD1 and SUP-B15 cells, it is thought that combinations of resveratrol with SKI II and PDMP suppress cell proliferation by inhibiting DNA synthesis through causing S phase arrest (Figure 3.2.1.a, b and Figure 3.2.2.a, b).
The effects of resveratrol or its combinations with different agents on cell cycle in different types of cancer have been shown in the literature [178]. Determined increased expression of p21 and p27 and decreased expression of cyclin A and D in T-ALL cells treated with resveratrol, which resulted in G0 / G1 phase arrest. In human colorectal cancer cells, resveratrol caused arrest in the S phase and its combination with 5-flurouracil increased S phase population as compared to resveratrol [160]. Resveratrol arrested OCM2 AML cells at S phase [169]. In natural killer cell (NK) lymphoma cells, resveratrol suppressed proliferation by arresting cells at G0 / G1 phase [200]. In B-ALL SUP-B15 cells, 75 μM resveratrol treatment resulted in S phase arrest and proliferation was suppressed [177]. In SUP-B15 cells, myriocin combinations did not have significant effect on the cell cycle (Figure 3.2.2.c) and supported cell proliferation results (Figure 3.1.4.c). The combination of myriocin with resveratrol supported the results of cell proliferation (Figure 3.2.1.c) by arresting SD1 cells at S phase (Figure 3.1.3.c). This study has shown for the first time how resveratrol together with targeting sphingolipid metabolism has an effect on the cell cycle and cell proliferation on the Ph + ALL model.
52